CN1296413A - Composition and method for modulating cytokine release in response to genotoxic agents - Google Patents

Abstract

A previously unknown mechanism for cytokine release in response to genotoxic agents, as well as therapeutic and diagnostic procedures and techniques which are based on that mechanism, are disclosed. The mechanism involves the recognition of damaged DNA by DNA-protein kinases and the subsequent phosphorylation of substrates that leads to cytokine release. Methods to modulate the activity of DNA-protein kinases and thus the release of cytokines in response to genotoxic agents are described. Assays for DNA-protein kinase activity which can be used to monitor such modulation are also disclosed.

Description

Adjusting responds to the compositions and the method for the release of cytokines of gene poisonous substance

Cross reference the application of relevant provisional application requires the U.S. Provisional Patent Application No.60/073 that submitted on February 4th, 1998,640 priority according to 35 USC § 119 (e).

Invention field

The present invention relates to cytokine and gene poisonous substance.More particularly, the present invention relates to the energy control example as regulating the compositions and the method for the release of cytokines of cell when responding to one or more gene poisonous substances of contact.

Background of invention

A. gene poisonous substance

This area is well-known, and the gene poisonous substance is can directly or indirectly cause or those chemical compounds of inducing DNA damage or handle as heat or radiation.This damage can cause sudden change, cell cycle stops and/or cell death.This damage can be at nucleic acid base or at sugar-phosphoric acid skeleton, perhaps can be the strand or the double-strand break of DNA chain.Sudden change is when taking place, and it is the hereditary change in the DNA sequence, or can cause the dna modification collection of illustrative plates of cell function hereditary change.

The gene poisonous substance is found in the environment and is the natural pollutant in natural constituents such as ultraviolet or ionizing radiation or the food such as aflatoxin or artificial pollution such as the benzopyrene in medicated cigarette or the industrial smog.The gene poisonous substance also can be used for pharmacy and healthy relevant purpose.For example, many anticancer radiation and chemotherapies are gene poisonous substances.Similarly, ultraviolet is the maximum gene poisonous substance of humans and animals contact, and it can be used for useful purpose such as process hides.In addition, light or ionizing radiation can be used with light activated drug regimen, are used for dermatological and anticancer therapy and are used for the blood sterilization.

To one of biological response of gene poisonous substance is stopping of cell cycle, thereby repairs time enough is provided for carrying out DNA.This reparation may also may get nowhere in success, and it depends on following factor: the level of the DNA repairase in the cell, the degree of DNA damage and type or the like.Stopping at of cell cycle prevents by playing an important role in the hereditary material acute injury that causes without the cell division of repairing.If damage can not be repaired, cell just starts apoptotic responses, i.e. the apoptosis approach.This can cause cell cycle to stop and/or apoptotic cells in the general process of molecular signal recently by P.Herrlich, C.Blattner, A.Kneber, K.Bender and H.Rahmsdorf are at " nuclear and the non-nuclear target of the gene poisonous substance of gene expression in inducing; the total principle in yeast, rodent, people and the plant ", Biolongical Chemistry, 378 volumes, the 1217-1229 page or leaf, 1997; With J.Y.J.Wang at " at the cell response of DNA damage ", " contemporary cytobiology comment ", 10 the volume, the 240-247 page or leaf, 1989 summarize.

B. the The Long-term Effect of gene poisonous substance ... the sudden change of DNA

The The Long-term Effect of gene poisonous substance is the sudden change among the DNA, and this occurs in DNA and repairs when getting nowhere.These sudden changes are heritable changes in the DNA sequence, and are Fundamentals in the human oncogenic process.And unclear from mutation fixation be the institute that forever is established to cancer development that changes of DNA in steps.A feature of most human cancers is to contact with the gene poisonous substance and arranged between time of cancer development secular, for many years incubation period.Thing really like this, although sudden change is just fixing rapidly behind contact gene poisonous substance.The ongoing change that is called as the initial tissue that contains mutant cell of tumor is subjected to the promotion of the inductive cytokine of gene poisonous substance, causes the appearance of tumor.

C. the short-term effect of gene poisonous substance ... the release of cytokine

The important short-term effect of gene poisonous substance in the animal and human is systematic and relates to tissue and reply immunosuppressant and other physiological effects that comprises erythema, inflammation, fever, antigen-specific.These influences by cytokine mediated (summary is seen T.Luger and T.Schwarz, " epithelial cell source cytokine ", Skin Immune System, J.D.Bos edits, CRC Press Inc., Boca Raton, Fla, 1990, pp257-291).The present invention and these consistent based on cytokine at replying of gene poisonous substance.

This area is well-known, and cytokine is big diversified protein family, and they are discharged by a cell, influence other cells and/or the activity of himself.The level of cytokine is subjected to the interferential adjusting of replying of cellular function usually, and is used for regulating and contact afterwards tissue, tract and whole organic replying and stable state with the gene poisonous substance.

Known cytokine is not single release, but discharges in colony's mode, thereby produces the characteristic list that a kind of gene poisonous substance is replied.For example, induce simultaneously by the sunburn that sunlight middle-ultraviolet lamp (gene poisonous substance) causes: (a) can cause the expression of the il-1 (IL-1) of having a fever, (b) can activate the interleukin-6 (IL-6) of liver function, (c) can induce the tumor necrosis factor (TNF α) of inflammation, it can cause the immunosuppressant of partial antigenic specificity and activate the virus of hiding, (d) IL-10 INTERLEUKIN-10 (IL-10) of energy suppressor inducer T lymphocyte, (e) the of short duration reduction of ICAIU 1 (ICAM-1) then increases, ICAM-1 controls lymphocytic infiltration, and (f) reduction of IFN-(IFN-γ), IFN-γ regulates immunne response and multiple other cellular elementss.

As for the extracellular signal, most important cytokine is TNF α and IL-1, because they can not only produce they self physiologic response, and can induce other remote cellular expression cytokines.Control is one of main purpose of the present invention by this extracellular signal based on cytokine that the gene poisonous substance produces.

The prevailing mechanism of cytokine effect is to discharge and move to other cells from cell by them.But in some occasion, a kind of cytokine can influence the extracellular signal by being illustrated in the damaging cells surface.Correspondingly, be used for herein, term " cytokine " discharges " and " cytokine generation " be used to comprise that cytokine shows the extracellular signal that a kind of cytokine produces from the actual release of cell with in the extracellular of cell surface.These terms also are used to comprise widely any cell mechanism that can cause the extracellular cytokine signaling to increase, and include but not limited to transportation in a kind of de novo synthesis, precursor processing, cell of cytokine, dissociate in the extracellular and surface display.

D. this area is to two fens theories of strictness of misunderstanding (1) mistake of gene poisonous substance mechanism of action

Until today, those skilled in the art thinks that still the damage of DNA causes stopping immediately of cell cycle, and the fixing or apoptosis that the latter can cause contacting the back dna mutation with the gene poisonous substance is replied.This principle by L.H.Hartwell and M.B.kastan in " cell cycle control and cancer ", " science " 266 volumes, the 1821-1828 page or leaf is introduced in 1994.

On the other hand, cytokine induces the result who is considered to cell membrane damage and produces, i.e. the result that the target damage of shifting out because of nucleus produces.This principle is rolled up at " nuclear factor к 6: the crucial transcription factor in the chronic inflammatory diseases " " New England Journal of Medicine " 336 by P.Bamer and M.Karin, introduces in the 1066-1071 page or leaf.

As discussion hereinafter, consistent with the present invention, have been found that this viewpoint, promptly between causing influence and film (kytoplasm) damage that (b) causes the extracellular signal of DNA (nuclear) damage of signal in the cell, (a) have the differentiation of strictness, and be incorrect.In fact, the DNA damage that causes of gene poisonous substance contact also can cause the extracellular signal by production of cytokines.

And, also consistent with the present invention, have been found that by this mechanism to produce the protein kinase that cytokine needs DNA.As a result, the cytokine that causes of gene poisonous substance contact produces and can control by the level and/or the activity of control DNA one protein kinase.This control mechanism is efficiently, but is not understood in the past and use.

(2) two fens theoretical evidences of the strictness that leads to errors

Think that traditionally inducing of cytokine only is the change because of cell membrane, the evidence of this viewpoint is very strong.Set up the approach of several protein activations that cause the expression of energy active cell factor gene.All these approach are all begun by the protein kinase that is positioned at epicyte, and the interior part of the most kytoplasms with a kind of transmembrane protein of these protein kinases is relevant, and the extracellular part of this transmembrane protein is a kind of cell receptor (Y.Devary, R.Gottlieb, T.Smeal and M.Karin are at " the mammal ultraviolet is replied by the Src tyrosine kinase and caused " " cell " 71 volumes, the 1081-1091 page or leaf, summary in 1992).

These protein kinases are activated by the incident on the cell membrane, leave nucleus, activate other kytoplasm kinases, and the latter is accumulated into the phosphorylation system of the gene activation factor such as AP-1 and NF к B.The DNA binding site of the gene activation factor of these modifications or release sees the promoter region of cytokine encoding gene usually.For example, a lot of this binding sites see the promoter sequence of TNF α gene, as S.Takashiba, L.Shapira, S.Amar and T.Van Dyke be at " clone of human TNF alpha promoter region and analysis " " gene " 131 volume, 307-308 page or leaf, the introduction in 1993.Therefore can suppose like this that these film activity factors pass through a kind of like this approach with the combining of binding site on its cytokine gene promoter region, promptly change the change that causes cytokine-expressing on the cell membrane.

Strict two minutes theoretical further evidences are from some nearest reports, and their proof gene poisonous substances for example UV can directly make cell surface receptor trimerization and activation energy start the kinases of the cascade reaction that can cause signal conduction and gene expression by inference.See I.Warmuth, H.Harth, M.Matsui, N.Wang and V.De Leo, " ultraviolet radiation is induced the phosphorylation of EGF-R ELISA " " cancer research " 54 volumes, 374-376 page or leaf, 1994; With C.Rosette and M.Karin, " ultraviolet and osmotic pressure: activate the JNK cascade reaction " " science " 274 volumes, 1194-1197 page or leaf, 1996 by multiple somatomedin and cytokine receptor.

But the strongest evidence from study system the most completely--release of cytokines after-ultraviolet (UV) radiation.See T.Schwarz and T.Luger, " influence that the UV radiation produces the epidermis cell factor " " Journal of Photochemistry and Photobiology, B:Biology " 4 volumes, 1-13 page or leaf, 1989.Many cytokines such as IL-1, IL-6, IL-10, TNF α, ICAM-1 and IFN γ known after UV handles in, the level of its gene expression (transcribing) and release can change.

Utilize this system, carried out some experiments, these experiments be considered to can (a) final certification place one's entire reliance upon incident on the cell membrane of gene activation, and (b) get rid of DNA as the activatory target of cytokine.See Y.Devary, C.Rosette, J.Didonato and M.KariN, " NF к B is by the UV-activated nuclear signal that do not rely on " " science " 261 volumes, the 1442-1445 page or leaf, 1993 and M.Simon, Y.Aragane, A.Schwarz, T.Luger and T.Schwarz, " UVB photoinduction nuclear factor к B (NF к B) does not rely on the chromosomal DNA damage in acellular Cytoplasm ", " Joumal of Investigative Dermatology " 102 volumes, the 422-427 page or leaf, 1994.

In these experiments of extensively being quoted, cell is by denuclearization (chemistry and physical treatment are to remove enucleation), the UV radiation of the cytosome of generation.Although nuclear (and genomic DNA) does not still detect the activation of genetic transcription enhancer NF к B.

Perhaps Just because of this, caused a common recognition widely among these experiments those skilled in the art, promptly the gene poisonous substance is handled inducing of cytokine gene expression of back and is not relied on DNA damage, and the incident on the cell membrane that places one's entire reliance upon.For example, G.Vilie, A.Tanew-Ilitschew and R.Tyrrell are at " oxidative pressure that the UVA radiation produces to human skin fibroblast in the activation of NF к B ", Photochemistry and Photobiology, 62 volumes, 463-468 page or leaf, 1995, in conclude: " still, the activation that the UVC radiation of NF к B in enucleate cell relies on is true, and the activation that the UVB radiation relies on is presented in the akaryote extract and takes place.Therefore at least for these two kinds of handled things, examine not participating in activation approach ".

Also exist some evidences to show, DNA damage may be the initiation event that the inducing cell factor gene is expressed.These report demonstration: the dose response relationship of cytokine gene expression becomes at lower gene TD at the transit cell of DNA repair-deficiency (sees B.Stein, H.Rahmsdorf. A.Steffen, M.Litfin and P.Herrlich, " the inductive DNA damage of UV is an intermediate steps in the UV abduction delivering of I type human immunodeficiency virus, collagenase c-fos and metallothionein " " molecule and cytobiology ", 9 volumes, the 5169-5181 page or leaf, 1989); With strengthen the DNA repair ability and can reduce cytokine-expressing and release (D.YAROSH by giving the foreign DNA repairase, L.Alas, J.Kibitel, A.O'Connor, F.Carrier and A.Fomace, " the cyclopropane pyrimidine dimer among the UV-DNA is induced the release of the solubility mediators that can activate human immunodeficiency virus's promoter ", Journal of InvestigativeDermatology, 100 volumes, 790-794 page or leaf, 1993).

But before the present invention, people do not recognize that the DNA damage that the gene poisonous substance produces can be transformed into the activatory biochemical mechanism of cytokine gene expression, and this is the shortcoming that is very easy to find among those skilled in the art.For example, Herrlich, Blattner, Knebel, Bender and Rahmsdorf write in " biochemistry ", above 1223 pages in 1997: " in sum, yeast is the same with mammalian cell, has the transcriptional control approach that DNA damage relies on.According to yeast genetics, its component has all obtained good description, but damage identification and signal is essential not clear ".Similarly, Stephen Jackson write at " DNA rely on protein kinase " " international biochemical and cytobiology magazine " 29 volume 935-938 pages or leaves the 937th page in 1997: " and; by elicitor protein tyrosine phosphorylation cascade reaction; the activation energy inducing cell DNA damage signal pathway of DNA-PK probably, the latter and transcribe, apoptosis and cell cycle mechanism is closely related.But the positive evidence that does not also have acquisition in the signal conduction, to work up to now, ".Even more recently the time, Jean Wang write in " contemporary cytobiology comment " (above) 242 pages in 1998: " UV replys and can be mediated by cytoplasmic membrane.Whether the inductive damage of UV (cyclobutane dimer and other photoinduction things) can produce the proteic signal of activation Rad3/ATM sample in mammalian cell be unknown." Herrlich and Rahmosdorf laboratory in 1998 at C.Blattner; Klaus Bender, repeated their viewpoint in 1997 in the 2832nd page in " Photoproducts in transcriptionally active DNAinduce signal transduction to the delayed U.V.-responsive genes forcollagenase and metallothionein " " oncogene " 16 volume 2827-2834 pages or leaves of Peter Herrlich and Hans Rahmsdorf: " do not know still how DNA damage comes the inducement signal transduction in the open gene.”

There is not a kind of biochemical mechanism---crucial connection, relative dna damage may be that the data of initiation event of cytokine gene expression are all out in the cold mostly.And, do not recognize this mechanism, the control of this mechanism is even the control of release of cytokines clearly is impossible.The key element that the present invention provides these to lack for this area.

The invention summary

According to top viewpoint, a target of the present invention provides at the method and composition that contacts control (adjusting) release of cytokines in back with the gene poisonous substance.

A concrete target of the present invention is to be ultraviolet when the gene poisonous substance---humans and animals provides such method and composition, this control (adjusting) to relate to during the gene poisonous substance of normal contact to reduce cytokine response in the release of this gene poisonous substance.

Further concrete target of the present invention is to provide such method and composition when being a kind of chemical treatment reagent that is used for treatment of cancer or radiotherapy reagent when the gene poisonous substance, and is same, controls (adjusting) and relates to the release that reduces cytokine.

A further target of the present invention is to make to accept organ transplantation and accepting immunosuppressive drug and reducing sensitivity to gene poisonous substance (for example ultraviolet radiation) with the individuality that prevents transplant rejection.

Another concrete target of the present invention is that such method and composition is provided when being a kind of immunosuppressant reagent when the gene poisonous substance, and control (adjustings) relates to the increase cytokine response in the release of this gene poisonous substance.

Another target of the present invention is determine to need to regulate the level of its DNA-protein kinase and/or active in to regulate the individuality of replying of this individuality to one or more gene poisonous substances.Target is relevant therewith, and a further target of the present invention is to determine its level and/or the active specific DNA-protein kinase that needs this adjusting.

A further target of the present invention provides determines a kind of immunosuppressant reagent whether with a kind of method of horizontal administration, and wherein this level can provide required reduction or the increase that is enough to influence the DNA-of the release of cytokines that responds to gene poisonous substance protein kinase activity.

Another target of the present invention provides the improvement test that detects the DNA-protein kinase activity.

The present invention finishes above-mentioned and other targets by following discovery: DNA-protein kinase, one group can discern change among the DNA such as double-strand break enzyme and can other albumen of phosphorylation and/or the enzyme of himself in the release of cytokines that responds to the gene poisonous substance, work.Specifically, have been found that transcribing and/or translate and needing of cytokine gene after one or more protein kinases are for contact gene poisonous substance.

Be used for herein, " DNA-protein kinase " is by other albumen of phosphorylation and/or himself comes albumen that the change of dna structure or conformation is replied and the member of albumen composition family.The feature of this enzyme family is by S.Jin, and S.Inoue and D.Weaver are in " function of the protein kinase of DNA dependence ", " cancer exploration ", 29 volumes, 221-261 page or leaf, summary in 1997.

Before this, these enzymes only are considered to participate in (1) immune competent cell, and especially those need relate to the formation of cell of the gene rearrangement of double-stranded DNA fracture, (2) regulation of Cell Cycle and apoptotic responses after the reparation of DNA double center chain fracture and (3) DNA damage.These contacts by M.Hoekstra " ATM protein kinase family is to replying that DNA damage and cell cycle checkpoint are regulated ", " modern genetics and auxology comment " 7 volumes, 170-175 page or leaf, summary in 1997.

Clearly, with regard to UV---with regard to the gene poisonous substance of extensive existence, not having the member in the DNA-protein kinase family, known to discharge cytokine relevant with the inductive cell of UV.As mentioned above, the viewpoint that those skilled in the art held, promptly cytokine gene expression is controlled by membrane interaction, makes these DNA-protein kinases can not be considered to participate in causing the signal transduction cascade reaction of release of cytokines.

For example, in the summary of quoting in the above, M.Hoekstra writes in the 170th page: " in this article, I have discussed the feature of PIK kinases (P13-kinases-related protein kinase) family as the pick off of Cycle Regulation." in another example; Hosoi etc. are " a kind of inhibitor of phosphatidylinositol 3-kinase---wortmannin induce the DNA of radioresistance synthetic and make cell to bleomycin and ionizing radiation sensitivity " at its title; " international journal of cancer " 78 volumes; 642-547 page or leaf; prove in 1998 the article; wortmannin is a kind of inhibitor of DNA-protein kinase, but the wortmannin pair cell factor the influential inhibition that all is summed up as membrane-bound phosphatidylinositol 3-kinase (PI-3).At the 645th page of their article, these authors write: " wortmannin suppresses by making P13 kinases inactivation suppress cytokine/chemokine mediated signal transduction path ... "

Being set out among Fig. 1 of this section background field represented with diagram, the box of its middle and upper part band point shows that DNA damage causes the activatory classical pathway of DNA-protein kinase, the protein substrate of this tyrosine phosphorylation key, and the latter causes because the cell cycle that signal produces in the cell stops and apoptosis.On the other hand, release of cytokines was summed up as the approach in the box of wearing down a little in the past in this area, wherein the gene poisonous substance influences cell membrane and/or cell-membrane receptor, the latter activates a kind of fat and protein kinase cascade reaction, and the latter causes cytokine gene expression and extracellular release of cytokines.

Fig. 2 has shown the gene poisonous substance response pathway consistent with the present invention.The main distinction of Fig. 1 and Fig. 2 is, the as broad as long early stage and late incident of the diagram of prior art among Fig. 1 promptly occurs in and contact 3 hours with the gene poisonous substance and contacted 6 hours or more late incident (late incident) with occurring in interior incident (incident in early days).It is an importance that forms the discovery on basis of the present invention that this time divides.

Among Fig. 2 among approach and Fig. 1 the prior difference of approach be, Fig. 2 comprises and production of cytokines is considered as one of influence that DNA damage is caused owing to the gene poisonous substance, and this generation has caused that following biological impact such as red class, antigen specific immune suppress, melanogen generates and be tanned.This cytokine of gene poisonous substance exerts an influence in the right lower quadrant demonstration of the right hand box of Fig. 2, and as pointed in this box, this influence depends on the activation of at least a DNA-protein kinase.

The dissimilar dissimilar DNA-protein kinase of DNA damage activation that the different genes poisonous substance produces.For example, the double-strand break activation is known as the DNA-protein kinase of DNA-PK and ATM usually, and UV inductive photoproduct activation in DNA is known as the DNA-protein kinase of FRAP usually.Ongoing research will be expected to differentiate the other forms of DNA damage that is suitable for this pattern and the member of DNA-protein kinase family in global different experiments chamber, and the present invention will can be applicable to these DNA damage/DNA-protein kinase components subsequently equally.

As illustrated in Fig. 2, the common aspect of institute of the present invention foundation is that (1) DNA-protein kinase is that the proteic phosphorylation in center and (2) downstream that identification DNA changes causes cytokine gene expression, and promptly cytokine gene transcribing and/or translating.Find that based on this cytokine that responds to the gene poisonous substance produces and can control by the level or the activity of regulating one or more DNA-protein kinases.

This adjusting can be finished with the following method: (1) uses one or more inhibitor to suppress the activity of one or more DNA-protein kinases, (2) improve or reduce gene transcription and/or the translation that participates in producing one or more DNA-protein kinases, and/or (3) are by such as provide higher level and the substrate by its phosphorylation of damage dna its reaction or higher level to improve the activity of one or more DNA-protein kinases to kinases.

In a kind of special applications of the present invention, the chemical compound such as the rapamycin that suppress the DNA-protein kinase are used to inducing of the blocking gene poisonous substance pair cell factor.The inhibitor of other DNA-protein kinases comprises pyrophosphoric acid, wortmannin, 6-dimethyl amine purine, Pyridione derivatives OK-1035 and single stranded DNA, roll up at " protein kinase that DNA relies on; DNA-PK:10 and endless observation " " biochemistry and cytobiology " 74 as S.P.Lees-Miller, the 503-512 page or leaf, the introduction in 1996.

Contact gene poisonous substance may be unexpected, for example contacts with environmental contaminants or daylight in daily life.Contact also may be have a mind to, be deleterious side effect, for example when the radiotherapy of autotelic sun tan or cancer or chemotherapy.Inducing of cytokine may be undesired, because they are inhibitive ability of immunity, struvite, activation virus, cause undesired chromogenesis, keloid, adhesion or cicatrix or other former of contact with the gene poisonous substance or a pair result.

The specific example of these aspects of the present invention be included in skin protection and sun care preparations and be used for preventing sunlight and the drug products of pollutant to the activation of the activation of for example red class of the undesired side effect of DNA damage, inflammation, immunosuppressant, the herpes infection of hiding, protease (for example collagenase and metallothionein) and skin carcinoma in mix one or more DNA-kinases inhibitors such as rapamycin or rapamycin sample chemical compound.

DNA-kinases inhibitor for example rapamycin or rapamycin sample chemical compound also can be used for uniting use with cancer chemotherapeutic drug or radiotheraping method, reduce and the relevant side effect of these treatments, for example fever, red class, feel sick, vomiting, headache, feel cold and the generation of undesired pigment.In of the present invention these were used, preferably at the close time administration that contacts with gene poisonous substance such as sunlight, air pollutants, chemotherapy or ionizing radiation, more preferably 30 minutes before contact were to administration in 1 hour for the DNA-kinases inhibitor.

The present invention also is used in the main harmful side effect that prevents immune transplantation therapy when transplanting.Organ transplantation has become very general after introducing extraordinary immunosuppressive compounds of toleration such as ciclosporin.But of this immunosuppressive therapy is main that side effect is to make patient increase the danger that skin carcinoma takes place when skin is exposed to sunlight.See M.Glover, C.Proby and I.Leigh, " skin carcinoma in the rectum transplant patient " " Cancer Bulletin " 45 volumes, 220-224 page or leaf, 1993.

Because it is to disturb calcineurin that ciclosporin provides immunosuppressant mechanism, it does not block the release of cytokines after the UV-B contact.See A.Marionnet, Y.Chardonnet, J.Viac and D.Schmitt, " horn cell culture normal and that transform is replied and excretory difference the il-1 and the tumor necrosis factor of Ciclosporin A and ultraviolet-B radiation " " experiment Dermatology " 6 volumes, the 22-28 page or leaf, 1997.Therefore, ciclosporin does not have DNA-kinases inhibitor such as rapamycin are blocked the UV-inducing cell factor when skin is exposed to sunlight beneficial effect.

The present invention has introduced following content, when keeping required immunosuppressive condition and carry out organ transplantation, in general, the organ of contact gene poisonous substance, particularly be exposed to a kind of DNA-kinases inhibitor of dermal application of sunlight such as rapamycin before contact gene poisonous substance, handle simultaneously or afterwards, with the cancer that prevents that the gene poisonous substance from causing.When the DNA-kinases inhibitor self is a kind of immunosuppressant (for example rapamycin) that can suppress transplant rejection, this inhibitor can with or not with the medicine of other immunosuppressant and/or other types or handle and use.In this case, consider that simultaneously ability that ability that it suppresses transplant rejection and its inhibition respond to the cytokine generation that the gene poisonous substance contacts selects the administration process and the dosage level of immunosuppressant.

Other application of the present invention are included in can increase the chemical compound (after this be called " DNA-protein kinase reinforcing agent ") of the activity of DNA-protein kinase with the increase cytokine induction after the gene poisonous substance is handled.Some gene poisonous substances is handled and is used to induction of immunity and suppresses to reply like this.For example, psoralen+light is used to skin transplantation and psoriasis comes the induction of immunity SC factor and inhibition antigenic specificity self to reply.The mechanism of listing in the comparison diagram 2, the chemical compound that can strengthen the DNA-protein kinase activity can make these immunosuppressive therapies rapider, strong and/or general, so improves the effect of treatment.Like this, according to the present invention, the chemical compound that can strengthen the DNA-protein kinase activity is used to simultaneously or substituent group is strengthened required immunosuppressant because of poisonous substance and replied.

For example, according to these aspects of the present invention, one or more can and thereby can stimulate the chemical compound of DNA-protein kinase activity to use when the gene poisonous substance is handled or substituent group is handled the release of immune stimulatory inhibition cytokine because of poisonous substance and alleviation to the autoimmune response relevant disease is provided as UV radiation DNA or short segmental double-stranded DNA effect.The example of this chemical compound comprises fat or the liposome that combines double-stranded DNA and/or its homologue.

Another application of the invention be identify its one or more DNA-protein kinases level and/active the needs regulate to regulate its individuality of replying (sensitivity) at one or more gene poisonous substances.According to these aspects of the present invention, screen the special DNA-protein kinase of determining to cause this patient symptom to suffering from patient's DNA-protein kinase activity and/or the level that produces the disease of not enough or excessive cytokine-expressing owing to a kind of gene poisonous substance.For example, the dermatitis of some type is because immune system causes environment UV or pollution overreaction as atopic dermatitis, lupus erythematosus and porphyria.By screening DNA-protein kinase activity in these patients, determined to obtain from special DNA-kinases inhibitor the patient of benefit.In of the present invention these are used, from tissue sample, prepare cell extract, and carry out the test of DNA-protein kinase.

For example, the type that this test as is hereinafter introduced among the embodiment 4, wherein antibody is used to immunoprecipitation DNA-protein kinase, then with dissimilar DNA damage and substrate such as the p53 albumen of sedimentary DNA-protein kinase contact.By measuring the degree of p53 phosphorylation, just can determine the activity of this DNA-protein kinase.When comparing with normal control, these researchs can determine whether that more DNA-protein kinase is expressed or this DNA-protein kinase activity in this tissue is not higher in diseased tissue.This information can be used for diagnosing the illness and selecting therapeutic treatment subsequently.

According to the various aspects of front of the present invention, the test that is used for detecting DNA-protein kinase levels/activity need determine for example, whether a kind of administration level of medicine is enough to suppress or the inducing cell factor discharges.As the elaboration among the embodiment 4 hereinafter, the invention provides effective test and be used for this purpose, wherein: (1) obtains cell sample from receptor, (2) from sample, obtain the prepared product that contains the DNA-protein kinase, (3) with the DNA damage of this prepared product and such kinases sensitivity with and suitable substrate one work, and (4) are used for measuring kinase whose levels/activity with the substrate phosphorylation level.

Aforementioned will connecting further with detailed description of the present invention and preferred embodiment thereof below with other aspects of the present invention goes through.

The accompanying drawing summary

Fig. 1 shows that this area is in the past to participating in the flow chart of cell to the understanding of gene poisonous substance response pathway.As shown here, the DNA damage of gene poisonous substance is considered to activated dna-protein kinase, and the latter then causes substrate phosphorylation, then the activated cell cycle stop with apoptotic cells in incident.

Fig. 2 shows the flow chart of participation cell according to the present invention to gene poisonous substance response pathway.As shown here, the gene poisonous substance is to damage activated dna-protein kinase of DNA, and the latter then makes substrate phosphorylation, and then the extracellular of the activated cell factor discharges.

Fig. 3 is the tree diagram of DNA-protein kinase family, and the relation of known lipid and DNA-protein kinase shows according to the homology of aminoacid sequence.Albumen is divided into and has lipid kinase activity and protein kinase activity.Its title and organizational form have all been shown for every kind of albumen.The XISHI bar is represented non-homogeneous district, and solid subunit such as Ku or the protein bound zone of FKBP that representative is little.Hollow thick bar is represented the kinase activity site areas, and vertical speckle zone is the carboxyl terminal that shows DNA-protein kinase homology.For every kind of albumen all shown amino acid whose number (under the known situation) with the similar percent (identical or conserved amino acid replaces) in the kinases district of the proteic aminoacid sequence of ATM.This figure paint from V.Zakian " the ATM related gene: they tell us about the human gene what? " " cell " 82 volumes, the 685-687 page or leaf, nineteen ninety-five, and S.Jin, S.Inoue and D.Weaver " function of the protein kinase that DNA relies on " " cancer exploration " 29 volumes, the 22l-261 page or leaf.

Fig. 4 is the Western trace of TNF α protein expression in the human keratinized cell.Cell 200J/m from human cell line HaCat ²Uv b radiation or handle with 1ug/ml LPS, and 37 ℃ of incubations 24 hours.The preparation extract, electrophoresis on 15% polyacrylamide gel goes to nitrocellulose filter, carries out trace with the antibody of anti-human TNF alpha, and exposes with the ECL chemiluminescence system.Swimming lane is (1) radiation, and (2) radiation also began to handle in radiation with the 2ng/ml rapamycin in preceding 30 minutes, and handle with LPS (3), and handle with LPS and rapamycin (4); (5) identify contrast with TNF α.

Fig. 5 is presented at and exists or be exposed to TNF α promoter inducing chloramphenicol acetyltransferase (CAT) behind the UV when not having various DNA-kinases inhibitor.The XP12BE cell lacks the nucleotide cutting to be repaired, and with this cell of TNFcat transgenic transfection, formation can be expressed the XPTNF2 cell line of CAT under TNF α promoter.This cell began to handle in radiation with the DNA-kinases inhibitor in preceding 30 minutes, was exposed to 100J/m then ²UV-B.After 18 hours, the preparation extract, and use fluorescence chloromycetin substrate to detect the CAT activity.Product is separated with thin layer chromatography, and develops the color with UV-A.Sample is (1) independent substrate; (2) untreated cell; (3) the radiating cell of UV; (4) cell of handling with UV radiation and rapamycin; (5) cell of handling with rapamycin separately; (6) untreated cell; (7) the radiating cell of UV; (8) cell of UV radiation and wortmannin treatment; (9) untreated cell; (10) the radiating cell of UV; (11) cell of UV radiation and D-82041 DEISENHOFEN processing; (12) cell of LPS processing; (13) cell of LPS and rapamycin processing.

Fig. 6 shows that LPS handles back TNF α promoter inducing chloramphenicol acetyltransferase (CAT).The XPTNF2 cell is handled by the introduction of Fig. 5, except replacing UV with being exposed to 1ug/ml LPS.The CAT activity is calculated from the product amount of the amount of albumen extract and formation in 30 minutes, and the latter is undertaken quantitatively by fluorescence thin layer chromatoplate computer image analysis.

Fig. 7 is p70 in the human keratinized cell ^S6KThe Western trace of phosphorylation.Cell 100J/m from human cell line HaCat ²Uv b radiation or handle with 1ug/ml LPS, and 37 ℃ of incubations 24 hours.The preparation extract, electrophoresis on 10% polyacrylamide gel goes to nitrocellulose filter, uses the p70 at the people ^S6KSerine and the antibody of threonine phosphorylation form carry out trace, then, use the ECL chemiluminescence system to expose with the two anti-effects that connected horseradish peroxidase.Swimming lane is: (1) not radiation, and (2) radiation, (3) radiation also began to handle in radiation with the 2ng/ml rapamycin in preceding 30 minutes, and (4) handle with LPS and handle with LPS and rapamycin (5).

Fig. 8 shows FRAP and the ATM kinase activity on the p53 peptide.By human keratinized cell is that HaCat prepares extract.With the antibody incubation of extract, will precipitate with the bonded antibody of Protein G sepharose 4B-kinases product by centrifugal with anti-FRAP (black bar) or ATM (grey bar).The bonded FRAP kinases of pearl is mixed with FKBP albumen, with FRAP and ATM all with peptide moiety incubation of p53 albumen.In this mixture, add various DNA and inhibitor, as shown in the figure.At 30 ℃ of incubations after 2 hours, with 8 times of product dilutions, add elisa plate, the proteic antibody that albumen of modifying with anti-phosphoserine and phosphothreonine are modified and alkali phosphatase two are anti-to develop the color together with nitrophenols phosphoric acid substrate.Contrast comprises phosphoserine and phosphothreonine bovine serum albumin.The albumen of phosphorylation is measured with the optical density at 405nm place.

Fig. 9 shows the dose response curve of different rapamycin concentration to the inhibition level of TNFcat expression.The CAT activity part is calculated by the introduction of Fig. 5 by the level of acetylizad chloromycetin, and the activity during with no rapamycin compares.

Above-mentioned figure is incorporated herein and as description of the present invention, each illustrative embodiment of the present invention and a concrete ingredient of narrating, they are used to explain principle of the present invention.Should be appreciated that these figure and explanation are only is indicative, is not limitation of the present invention.

Detailed Description Of The Invention and preferred embodiment thereof

As discussed above, critical aspects of the present invention is: (1) finds that the DNA-protein kinase plays the role of a nucleus in the release of cytokines that responds to the gene poisonous substance and (2) are used this and found by administration DNA-kinases inhibitor (if release of cytokines will be lowered) or reinforcing agent (if release of cytokines will be increased) and regulate release of cytokines.

The A.DNA-protein kinase

The DNA-protein kinase begins to be to be realized in active animal and human's mutant of a member who lacks this enzyme family.

The mice that suffers from SCID (serious combined immunodeficiency disease) can not produce complete immune system owing to the immunoglobulin gene rearrangement defective.These mices are found has a kind of genetic mutation, and this sudden change makes a kind of important DNA-protein kinase activity inactivation in the immunoglobulin rearrangement process that relates to the double-stranded DNA fracture.In this way, the fracture of the developmental pilot process of immunocyte, double-stranded DNA is similar to DNA damage.See summary " protein kinase that DNA relies on " " international biochemistry and cytobiology magazine " 29 volumes of S.Jackson, 935-938 page or leaf, 1997.

The patient who suffers from genetic diseases AT (asynergy-capillary dilation) has muscle control forfeiture (ataxia) and a unusual blood vessel (telangiectasia) and easily suffers from hematologic cancers such as lymphoma.These patients have the aberrant gene that is called as ATM (AT sudden change).

After the SCID of these diseases and ATM gene are analyzed by clone and their nucleotide sequence respectively, they show with 3'-phosphatidylinositols (3'-IP) kinases to have very high homology, this kinases is a lipid family rather than proteic kinases, as S.Jackson, and summary above.This makes the people confuse when beginning very much, because there is not the lipid kinase activity to detect with biochemical route.People recognize that although sequence and 3'-IP kinases homology, these enzymes are protein kinase really now.

The DNA-protein kinase is found in all the eucaryon organisms from the yeast to people.Each cell has the DNA-protein kinase that more than one types belong to the family of this huge similar enzyme.The family of the aminoacid sequence relevant with the DNA-protein kinase comprises the original DNA-PK relevant with SCID sudden change, ATM, ATR, TEL1, MEC1, MEI41, FRAP, TOR1, TOR2 and RAD3 _CsWith Ku subunit and other, as Jin, Inoue and Weaver summary, above in 1997.This DNA-protein kinase family and sequence homology thereof are shown in Fig. 3.Studying to determine the other member of this family.

The DNA-protein kinase generally comprises two subunits, and one of them is more much bigger than another.Less subunit does not show in Fig. 3.SCID and AT disease are all produced by the sudden change of the gene of the less subunit of coding.The inhibitor of these genetic mutations and energy blocking dna-protein kinase activity has been used to the function that analyzing DNA-protein kinase has.

The B.DNA-kinases inhibitor

As discussed above, according to this aspect, the present invention relates to disturb the chemical compound of one or more DNA-protein kinase activities, no matter be avtive spot or a plurality of sites inactivation by directly making the DNA-protein kinase, still by directly disturbing the DNA-protein kinase to combine with the substrate of DNA and/or DNA-protein kinase phosphorylation, or by combining DNA and/or substrate with DNA-protein kinase competition, or the assembling of the subunit by disturbing the DNA-protein kinase.

The example that can be used for the DNA-kinases inhibitor of practice of the present invention comprises rapamycin, pyrophosphoric acid, wortmannin, 6-dimethyl amine purine, OK-1035, D-82041 DEISENHOFEN and single stranded DNA.These inhibitor can be used for various forms receptor being carried out administration with the known suitable carrier combinations of the present invention the time, comprise oral, injection and local the use.The level of administration will depend on special receptor, inhibitor and gene poisonous substance, and can measure according to the SMP of being used for the treatment of property processing.

Particularly, selecting the level of inhibitor administration that it can and can be reached by patient's tolerance is enough to reduce the DNA-protein kinase activity and reduces the expression levels that cytokine depends on one or more gene poisonous substances.The cytokine-expressing level can use standard technique known in the art to measure.Like this when dosage/route of administration not simultaneously, suitable inhibitor dosage level and administering mode can be selected by the expression that the monitoring cytokine depends on the gene poisonous substance.For example, the level that can carry out TNF α behind the contact UV to the receptor of having accepted one or more DNA-kinases inhibitors is monitored.Should be pointed out that secondary cytokine may have more complicated kinetics when originally cytokine such as TNF α increase behind contact gene poisonous substance, for example respond to gene poisonous substance such as UV, the level of interferon gamma may reduce, and the level of ICAM-1 may reduce, and then raises.If a kind of like this secondary cell factor is used to determine the dosage/administering mode of DNA-kinases inhibitor, these more complicated kinetics need be considered, for example, a kind of inhibitor level that can select is after the contact of gene poisonous substance the active horizontal dimension of interferon gamma to be held on the value of a prediction.

In particular preferred embodiment of the present invention, the level/mode of inhibitor administration is selected by the activity of monitoring of DNA-protein kinase, for example by using test discussed here to come monitoring of DNA-protein kinase activity.According to these embodiments, the level/mode of inhibitor administration is selected to make the activity of one or more DNA-protein kinases to produce a substantial reduction (for example 10% reduction, preferred 50%).For example, the active level of FRAP can monitoredly reduce the release of cytokine when contacting one or more gene poisonous substances with selection.Equally, UV contact can be the gene poisonous substance, at this moment selects the level of FRAP to reduce the release of TNF α.

The DNA-kinases inhibitor can be continuously or preferably administration when contact patients gene poisonous substance.More preferably, the DNA-kinases inhibitor with administration before the gene poisonous substance contacts, for example preceding 30 minutes in contact, administration when contact, carry out continuously and after a period of time, for example contact continuation in back 24 hours.Lacking in practice of the present invention passablely than administration in forward and backward, the process of contact, but is not preferred yet.C. rapamycin

The inhibitor of a kind of particular importance of DNA-protein kinase activity is a rapamycin.This inhibitor suppresses the assembling of the big small subunit of FRAP specifically, as E.Brown, and P.Beal, C.Keith, J.Chen, the introduction of T.Shin and S.Schreiber " the FRAP kinase activity is in vivo to the kinase whose control of p70S6 ", " nature " 277 volumes, 441-446 page or leaf, nineteen ninety-five.These authors have identified the sudden change FRAP that anti-rapamycin resistance can be provided, and have proved the specificity of this medicine.

Relative with rapamycin, wortmannin is the inhibitor of the important DNA-protein kinase activity of another kind, it blocks FRAP by changing the key amino acid that participates in phosphorylation, as M.Wymann, G.Bulgarelli-Leva, M.Zvelebil, L.Pirola, B.Vanhaesebroeck, M.Waterfield and G.Panayotou are in " wortmannin makes phosphatidylinositols 3 kinases inactivations by covalent modification lys-802; lys-802 is a residue that participates in the phosphoric acid transfer reaction " " molecule and cytobiology " 16 volumes, 1722-1733 page or leaf, the introduction in 1996.

Rapamycin is used to suppress immune system today clinically in migration process, because it has blocked the propagation of traditional immunocyte such as T cell, rolled up 651-657 pages or leaves 1997 years at " immunoregulation medicament: the manufacturing basis that in organ transplantation, uses " " PediatricNephrology " 11 as M.Suthanthiran and T Strom.It also is not used to use with short term contact gene poisonous substance, but has been used for life-time service to keep graft.Clearly, the patient who accepts immunosuppressant therapy comprises that the patient who accepts rapamycin is clearly indicated and will avoid contacting gene poisonous substance such as sunlight that in the All Time of immunosuppressant therapy this time may and can last for several years usually.See M.Glover, C.Proby and I.Leigh, " skin carcinoma in the rectum transplant patient " " Cancer Bulletin " 45 volume 220-224 pages or leaves, 1993.

At present, rapamycin is just in the latter stage of clinical trial, and by the title commodity production of Wyeth-AyerstLaboratories with Rapamune_, this laboratory is the branch of AmericanHome Products.It can use separately or with the ciclosporin of low dosage during transplant patient in treatment.

When by oral route was carried out administration, conventional rapamycin dosage range was that every day 2 is to 5mg.When local administration, local concentration is the scope of 0.2%w/v.When by the intravenous route administration, maximum tolerated dose is the scope of per kilogram of body weight 25mg.The dosage of immunosuppressant purpose clinically, is every square metre of skin scope of 0.5 to 25mg every day.

Specifically, every m ²The using dosage of about 21 to 25mg the rapamycin of body surface area begins through intravenously administrable, as C.G.Groth, C.Brattstrom and L.Backman are at " new trend in the implantology: how to utilize the sirolimus potentiality in clinical transplantation " " transplanting progress " 30 volumes, 4064-4065, in 1998 and B.Kahan roll up " rapamycin: the individual algorithm of the use of the rectum allograft receptors of handling based on 250 examples " " transplanting progress " 30,2185-2188, the introduction in 1998.Then by about 1-7mg/m ²The dosage of adjusting comes the low concentration that produces about 10-15ng/ml 2-3 the middle of the month in whole blood subsequently.But, most rapamycins concentrate in the erythrocyte, the blood plasma of rapamycin and tissue concentration and metabolism are less than 2 ng/ml, as D.Trepanier, H.Gallant, D.Legatt and R.Yatscoff be at " rapamycin: the research of distribution, pharmacokinetics and therapeutic domain: up-to-date " " clinical biochemistry " 31 volume, 345-351 page or leaf, the introduction in 1998.

Clearly, this tissue concentration is not enough to regulate the DNA-protein kinase activity behind contact gene poisonous substance and responds to this release of cytokines that contacts with regulating.This is to make the DNA-protein kinase basic inactivation of FRAP kinases specifically because need be higher than the concentration of 2ug/ml (or about 2nM).Shown in following embodiment 5, UV induces TNF α insensitive to the rapamycin that dosage is less than 2ug/ml.Therefore, according to the present invention, the rapamycin administration of using at present that makes whole blood reach the 10-30ug/ml level can not reach the rapamycin level that is enough to make the FRAP inactivation in blood plasma or tissue.

Until today, the recommended dose of rapamycin only is to measure by its ability that prevents the repulsion of transplant organ, is not to suppress the active required dosage of FRAP according to it in the non-immunocyte of gene poisonous substance contact to determine.In addition, the secular use that this dosage design is used to prolong not is to be used for regulating contacting with the gene poisonous substance.

Relative with these purposes in the past of rapamycin, according to the present invention, rapamycin is used to by with before gene poisonous substance such as UV contact and administration in the short time afterwards, reduce the release of inhibitive ability of immunity cytokine such as IL-1, IL-6, IL-10, ICAM-1 and TNF α, and, rapamycin uses to be enough to the reaching high-caliber dosage that reduces release of cytokines, promptly uses than being used for preventing the level that transplant rejection is high.Therefore, according to the present invention, rapamycin is used to protection rather than suppresses immune system, specifically, is to protect immune system behind contact gene poisonous substance.

Use a kind of immunosuppressive drug to protect immune thinking clearly against intuition and opposite with practice with the knowledge in existing field.D.DNA-protein kinase reinforcing agent

Though the prevailing application of the present invention relates to the minimizing of release of cytokines, in some occasion, need to increase this release, particularly, some therapy relates to be used the gene poisonous substance to handle to eliminate antigen-specific immune response (seeing below).

The reinforcing agent of DNA-protein kinase activity comprises the short-movie section (for example having less than the fragment of 25 kilobase to length) of (1) double-stranded DNA, promptly have can with the fragment of the DNA of the bonded end of DNA-protein kinase, (2) Sun Shang double-stranded DNA, this DNA can be long-chain or short chain, (3) situation about having been used up at the subunit of DNA-protein kinase is this subunit, (4) substrate of DNA-protein kinase phosphorylation and (5) ATP.

DNA-protein kinase reinforcing agent can carry out administration by the mode identical with the DNA-kinases inhibitor discussed above; except following content; therefore because reinforcing agent is bioactive, should uses the biological activity that to protect reinforcing agent and it is delivered to the supporting agent/carrier of target tissue.For example, in proteinic occasion, topical can use a kind of liposome transmission system to carry out administration.See Yarosh, U.S. Patent No. 5,190,762.

The similarity method of above the dosage/mode of reinforcing agent can be used inhibitor being discussed is measured.Therefore, reinforcing agent usually with administration before the gene poisonous substance contacts, administration continues in contact process, and continuity a period of time.Equally, the monitoring of cytokine and/or DNA-protein kinase level can be used to be identified for the suitable dose and the mode of concrete receptor, reinforcing agent and gene poisonous substance.E. the adjusting of release of cytokines

As discussed above, according to the present invention, the release of cytokines after the contact of gene poisonous substance can reduce or increase by the method for inhibition or enhancing DNA-protein kinase activity.In most occasions, the minimizing of release of cytokines is required, and prevailing example is the release of cytokines that reduces as UV contact result.

Another example that need reduce is relevant with the chemotherapy and radiation of cancer.Many chemotherapeutics all are genotoxicity as carmustine and ametycin and a lot of radiotherapy as handling with the X-ray.According to the present invention, give the side effect that one or more DNA-kinases inhibitors reduce treatment to the patient who carries out such treatment.

For other application of the present invention, one or more inhibitor are administration before a course of treatment preferably, and the administration process continued and continue a period of time after this course of treatment, for example 1 day etc.The DNA-kinases inhibitor preferably transmits in such a way, and they will arrive and be subjected to undesired genotoxicity injured tissues in treatment.In the chemotherapy occasion, the DNA-kinases inhibitor is usually with the identical mode administration of chemotherapy agents, but they also can be specifically at the modal position of chemotherapy side effect, for example the site (part or subcutaneous administration) of digestive tract (oral administration), scalp (topical) and/or chemotherapy agents injection.The special administration of same type also can be used for the occasion of radiotherapy.

Experiencing the protection that the patient of transplant rejection treatment need resist the gene poisonous substance especially, because their immune system has been subjected to inhibition.As discussed above, these patients suffer from cancer being exposed on the skin of sunlight usually, and the outbreak of this cancer is usually within several years of begin treatment.Though DNA-kinases inhibitor specifically rapamycin is used to this transplant rejection therapy, these use the protection that does not reach the contact of antagonism genotoxicity.The present invention introduces following content, and the DNA-kinases inhibitor should fields of implantation is multi-form to be transmitted with form and method method to be used to them.

Specifically, these inhibitor for example rapamycin should be before genotoxicity contact and the short period after the contact carry out administration.System at immunosuppressive drug uses owing to toxicity is restricted occasion, and the other local application of DNA-kinases inhibitor will be used to alleviate the influence of genotoxicity contact.Specifically, be exposed to the occasion of sunlight, one or more inhibitor will be used to be exposed to the skin area of sunlight by the part.Dosage will be adjusted to the release of cytokines that can suppress as the result of DNA damage, this means that the dosage level of one or more DNA-kinases inhibitors such as rapamycin will be than the other times height of transplant rejection therapy in the short time before and after the contact genotoxicity.

In some occasion, according to the present invention, being used to one or more as one or more transplant rejection medicines of DNA-kinases inhibitor such as rapamycin or its analog (for example SDZ RAD) is not that the transplant rejection medicine of DNA-kinases inhibitor such as Ciclosporin A or ascosin use.Because this medicine of two types does not generally have eclipsed toxicity, this coupling can allow bigger motility, reach the immunosuppressant purpose, and allow for the cytokine generation that patient provides the result of one or more gene poisonous substances of antagonism conduct contact simultaneously.

According to these aspects of the present invention, as the transplant rejection medicine of DNA-kinases inhibitor by can provide some immunosuppressant levels to use at least, the more important thing is, use with the level and/or the mode of the release of cytokines that can suppress to respond to the gene poisonous substance.The respective numbers of two types immunosuppressive drug can according to patient to the sensitivity of medicine and when reaching the protection level of required release of cytokines the quantity of required DNA-kinases inhibitor determine for each patient.

Though prevailing application of the present invention is relevant with the reduction production of cytokines, in some occasion, the present invention can be used for strengthening the cytokine that responds to the gene poisonous substance and produces.Specifically, the present invention can be used for treating the multiple disease that responds to the immune suppressor genes poisonous substance, comprises autoimmune disease.

For example, strengthen the cytokine generation and can be used for treating psoriasis, this is a kind of disease that is characterized as excessive hypertrophy of horn cell and T cellular infiltration skin.Two kinds of gene poisonous substances that are usually used in treating this disease are coal tar and psoralen+light.Every kind of occasion, the genotoxicity treatment all causes the activatory release of cytokines of energy suppressor T cell, and thereby alleviation disease symptoms.According to the present invention, DNA-protein kinase reinforcing agent is used to increase the release of cytokines level that responds to the gene poisonous substance.

Specifically, one or more DNA-protein kinase reinforcing agents give patient just before the administration inhibitive ability of immunity gene poisonous substance or preferably in administration the time.Use these reinforcing agents can allow to reduce the quantity of the gene poisonous substance of required administration, and in some occasion, reinforcing agent separately or only just can reach the release of the required immunosuppressant cell factor with a small amount of gene poisonous substance.

Other diseases that can handle in this way comprise atopic dermatitis, lupus erythematosus, arthritis and porphyria.These diseases are all passed through the T cell activation for every kind and are produced inflammation.This T cell activation can suppress by the suitable immunosuppressant cell factor.And DNA-protein kinase reinforcing agent of the present invention can increase the formation of those immunosuppressant cell factors when contact gene poisonous substance.

The hydraulic test of F.DNA-protein kinase activity

Following discovery, be that the DNA-protein kinase is that core between gene poisonous substance and the release of cytokines is got in touch biology, can make those kinases as (1) individual to the gene poisonous substance sensitivity and the test point (terminal point biology) of the effectiveness of the regulator of (2) release of cytokines.

Therefore, by measuring the level of the DNA-protein kinase activity of body one by one, we can determine individual level to the sensitivity that can produce the gene poisonous substance that DNA damage that this DNA-protein kinase replys replys.For example, for measuring the sensitivity to UV, we can measure the active level of FRAP, and are the sensitivity of measuring ionizing radiation (X ray), and we can measure the active level of ATM.The high-caliber DNA-protein kinase level that measures shows that this individuality will reply this reagent with low-level release of cytokines.

Both of these case all is undesired, and its evaluation all allows to carry out suitable diagnosis and/or therapeutic process.Specifically, for the individuality with high-caliber special DNA-protein kinase activity, the inhibitor of one or more these DNA-protein kinases can be used to regulate the cytokine response to the gene poisonous substance relevant with this kinases of this individuality by top introduction.For individuality with low-level DNA-protein kinase activity, carry out further diagnostic procedure and determine this low-level active source, for example can carry out a kind of genetic screening.A kind of like this DNA-protein kinase test can be to screen the basis is provided, if there is not this test, can't screen.

Effectiveness as for the regulator of release of cytokines, by carrying out a kind of test of DNA-protein kinase activity, we can determine whether that the DNA-kinases inhibitor of sufficient amount or reinforcing agent have given patient, and the adjustment of dosage, transfer mode or transmission scheme can adjust to suitable.

Using a kind of immunosuppressant reagent to experience immunosuppressant during the course of treatment patient, can use the test that detects the DNA-protein kinase activity to obtain required immunosuppressant level as DNA-kinases inhibitor such as rapamycin.Specifically, because the complicated metabolism and the bio distribution of immunosuppressant such as rapamycin, the blood levels of parent compound may not represented the biology effect of parent compound and metabolite thereof.The biology effect of this chemical compound and any metabolite thereof more can be directly monitored in the test that detects the DNA-protein kinase activity.

The test that can be used for the detection DNA-protein kinase activity level of practice of the present invention comprise those use radiolabeled ATP substrate as ³²The test that p-ATP, peptide substrates, gel electrophoresis and autoradiography show.Be seen in as D.Price J.Grove, V.Calvo, J.Avruch and B.Bierer, " inhibition of the inductive 70 KD S6 protein kinases of rapamycin ", " science " 257 volumes, 973-977 page or leaf, 1992.

Following Fig. 4 has introduced and a kind ofly can avoid using the radioreaction agent and can handle optimization test than the more sample of method of Price etc. simultaneously.When being used to measure the DNA-protein kinase level of a receptor, this test may further comprise the steps: (1) obtains sample cell from receptor, (2) use anti-DNA-protein kinase antibody to obtain to contain the kinase whose prepared product of DNA from sample, (3) with DNA damage type and this kinase whose suitable substrates effect of this prepared product and this kinases sensitivity, and (4) are used for measuring this kinase whose levels/activity with the phosphorylation level of substrate.The level of substrate phosphorylation uses specificity at being carried out quantitatively with the ELISA method of standard by the antibody of the substrate of this DNA-protein kinase phosphorylation in process of the test.

The specificity of this test and sensitivity can by change or make up anti-DNA-protein kinase antibody and/or test in the type and/or the concentration of the substrate that uses make amendment.Specifically, to the level of multiple kinase whose DNA-protein kinase activity, can be respectively by mixture simultaneous determination at step (2) and (3) anti-DNA-protein kinase antibody of formation and substrate.The sensitivity of this test can be revised by the concentration and/or the response time that increase antibody and/or substrate.

G. therapeutic combination

DNA-kinases inhibitor of the present invention can be prepared with suitable supporting agent separately, and perhaps they can make up mutually and/or with the gene protection agent of other medicinal ingredients such as non-DNA-kinases inhibitor.One of them such example is the compositions of rapamycin and one or more opacifiers such as titanium dioxide and/or one or more DNA repairases such as T4 endonuclease V in the local ingredients, uses before the UV in one or more gene poisonous substances of contact such as sunlight, in the process or afterwards.In the occasion of DNA repairase, it is a kind of preferred medication that the DNA repairase is wrapped in the liposome.See Yarosh, United States Patent (USP) 5,077,211,5,296,231 and 5,272,079.

In such ingredients, the level of DNA-kinases inhibitor is selected by the method for introducing above, for example uses a kind of DNA-protein kinase activity test.Level when the level of other active component in the ingredients generally is equivalent to this composition oneself use.

Experience the occasion of chemotherapy patient, DNA-kinases inhibitor of the present invention for example wortmannin can make up with a kind of genotoxicity chemotherapy agents such as ametycin and administration simultaneously.

DNA-protein kinase reinforcing agent of the present invention also can be prepared with suitable supporting agent separately, and perhaps they can for example be prepared with the gene poisonous substance mutually and/or with the medicament of non-DNA-protein kinase reinforcing agent.Example is that DNA, psoralen and a kind of suitable supporting agent combined administration of damage is in psoriasis patient's skin.

In order not to be subjected to the restriction of any way, the present invention will do more fully to introduce with the following examples.Be the materials and methods that each embodiment uses always below.The materials and methods cell culture

People's immortality HaCat cell line is from Dr.Jonathan Garlick, the State University of New York of Stony Brook.XPTNF2 cell line is according to J.Kibitel, V.Hejmadi, L.Alas, A.O'Connor, B.Sutherland and D.Yarosh are at " expression of the UV-DNA damage induced tumor necrosin ﹠ in mice and the people's cell " " Photochemistry andPhotobiology " 67 volume 541-546 pages or leaves, pCA is used in introduction in 1998 _TNFThe fibroblast XP12BE that the SV-40 of transfection XP family transforms prepares.This cell line is by mice TNF α promoter expression chloramphenicol acetyl transferasegene.These cell transformed tie up to have been added in 10% new-born calf serum and the antibiotic Dulbecco's modified Eagle's culture medium at 37 ℃ at moistening 5%CO ₂Cultivate in the incubator.The human keratinized cell of non-conversion is available from CloneticsCorporation, San Diego, California, and in having added the no albumen horn cell culture medium of cell at 37 ℃ at moistening 5%CO ₂Grow in the incubator.Medicine and genotoxicity are handled

Rapamycin, wortmannin, D-82041 DEISENHOFEN and lipopolysaccharide (LPS) are from SigmaChemical Company.These medicines are prepared by 1000 times concentration, and dilute with culture medium before use.The cell that rapamycin, wortmannin or D-82041 DEISENHOFEN are handled pretreatment 30 minutes and radiation post processing 18 hours before the UV radiation.Handle for LPS, LPS is joined in the cell handled 1 hour at 37 ℃, cell was with fresh culture feed again 18 hours then.

UV does not have the daylight lamp of optical filtering with 3.2J/m by Westinghouse FS-40 ²/ sec provides.For carrying out the UV radiation, culture medium to be removed, irradiated cell is used identical culture medium feed 18 hours again then.Cell extract and Western trace

Normal people's epithelium horn cell is cultured to 90% and connects remittance in the 10-cm plate, handle with medicine and gene poisonous substance then.Behind the incubation, with containing the standard electrophoretic buffer collecting cell of dodecyl sodium sulfate (SDS), with 70% power ultrasonic 2 seconds, in water, boiled 5 minutes ,-70 ℃ of storages with a Heat Systems Ultrasonic Sonicator.

For carrying out the Western trace, the sample buffer that cell extract and the 5ul of 10ug is contained dyestuff mixes, and with mixture boiled 2 minutes, and is splined on the SDS-PAGE gel (Biorad mini Protean II apparatus) of standard with collection layer buffer.For carrying out p70 ^S6KWestern uses 10% SDS-PAGE, for carrying out TNF α Western, uses 15% SDS-PAGE.Gel is undertaken by every gel 25mAmps, arrives the bottom of gel until dyestuff.A Semi-phor device (Hoefer Scientific Instruments, San Francisco, California) in 0.83mAmps/cm ²Electricity transfer printing 45 minutes is transferred to albumen on the Immobilon P film.Trace seals with 5% defatted milk powder, and resists 4 ℃ of combinations with one and to spend the night.For p70 ^S6KTrace, antibody is at the form (New England Biolabs) in threonine 421 and serine 424 phosphorylations.Wash trace then, and with the anti-rabbit antibody incubation of horseradish peroxidase.Trace develops the color with ECL test kit (Amersham), and exposes with HyperfilmECL.For TNF α trace, the monoclonal antibody of anti-human TNF alpha is from BoehringerMannheim Biochemicals.Wash trace then, and with coupling the sheep anti-mouse igg incubation of biotin, then and streptavidin-horseradish peroxidase incubation, then develop the color with ECL by top introduction.The TNFcat test

CAT test is undertaken by the introduction of Kibitel etc. 1998.In brief, the XPTNF2 cell is handled incubation 18-24 hour with medicine and gene poisonous substance.By three-wheel freeze thawing and centrifugal preparation extract, with every kind of extract of 50ug and the BODIPY-chloromycetin of 5noml (MolecularProbes, Eugene, Oregon) and the S-acetyl-coenzyme-A (Sigma ChemicalCompany) of 0.5mM mix.At 37 ℃ of incubations after 30 minutes,, and analyze with thin layer chromatography with ice-cold ethyl acetate extractive reaction product.Observe fluorogenic substrate and acetylate with UV-A, analyze the part that digital image calculates acetylation chloromycetin with computer image analysis technique, and thereby analysis CAT activity.Immunoprecipitation and the test of DNA-protein kinase activity

The HaCat cell grows in the culture dish of 10cm near connecting and converges.Then with them with Heat Systems Ultrasonic microtip with 70% power at TGN buffer (50mMTris, pH7.5, the 50mM phosphoglycerol, 150mM NaCl, 10% glycerol, 1%Tween20,1mM DTT, 0.5ul/ml Sigma P8340 protease inhibitor cocktail, 2nMmicroclystin LR) in ultrasonic 10 second.After centrifugal, extract is adjusted to lmg/ml albumen, is stored in-70 ℃ with identical buffer.For detecting the DNA-protein kinase activity, with the HaCat extract of 1mg 4 ℃ with the goat-anti FRAP of 2ug or anti-ATM antibody (Santa CruzBiotechnology) incubation 2 hours, mix with the Protein G PLUS-agarose (Santa Cruz Biotechnology) of 40 ul then, and spend the night 4 ℃ of stirrings.

Centrifugal collection sepharose 4B with the washing of TGN buffer, then with high-salt buffer (100mMTris, pH7.4,500mM LiCl) washing, is used PK buffer (25mMHEPES-KOH, pH7.9,50mM KCl, 10mM MgCl then ₂, 1mM DTT, 0.5ul/mlSigma P8340 protease inhibitor cocktail, 2nM microclystin LR, 200uMATP) washing, and be resuspended in 50ul PK buffer.The reaction modification comprise 0.5ug λ-DNA or with the G15T germicidal lamp with 250J.m ²The radiating λ-DNA of UV-C and the rapamycin of 40 ng/ml.Be initial action,, then the p53 peptide (amino acid/11-393, Santa CruzBiotechnology) of 50ug and the ATP of 1nmol added all DNA-protein kinase reactions FKBP (Sigma Chemical Company) the adding FRAP reaction of 2.6ug.At 30 ℃ of incubations after 2 hours, by centrifugal from sepharose 4B reaction product isolated.

Product is cushioned liquid (1.9g/L Na at the standard ELISA bag ₂CO ₃, 2.93g/LNaHCO ₃, the 0.1g/L thimerosal) and 8 times of dilutions, and get 200ul and place Immulon 2HB 96 hole elisa plates (Dynex Technologies Inc., Chantilly in hole Virginia), are incubated overnight at 4 ℃.Washing hole, and with 5% bovine serum albumin room temperature sealing 60 minutes.Hole and 10ml TBS/NonI (25ml/L 2M Tris pH8 then, 30ml/L 5M NaCl, 0.1%Nonidet P-40) the 2.5ul coupling in the anti-phosphoserine-BSA antibody of biotin and 2.5ul coupling the mixture of antibody (Sigma ChemicalCompany) of anti-phosphothreonine-BSA of biotin room temperature incubation 60 minutes.Washing hole, and with the 10mlTBS/NonI of 100ul in 10ul streptavidin-alkali phosphatase (Sigma Chemical Company) mixture room temperature incubation 60 minutes.Wash plate with the phosphoric acid Nitrobenzol phosphoric acid colour developing among the 0.1M diethanolamine pH10, uses the microplate detector 405 _NmReading.

Embodiment 1

Present embodiment has proved that after the UV contact, normal human keratinized cell is expressed TNF α, and this expression is suppressed by rapamycin.

From the 200J/m of the human keratinized cell of HaCat cell line from the FS40 daylight lamp ²Uv b radiation.Parallel culture was handled in radiation with 2ng/ml with rapamycin in preceding 30 minutes and 60 minutes afterwards.At 37 ℃ of incubations after 24 hours, with 70% peak power they are carried out 2 seconds of supersound process from these cell preparation extracts by they being scraped into phosphate buffer and with the little ultrasonic head of a Ultrasound Instrument (Heat SysTems Ultrasonicator).

Albumen with 10 micrograms is splined on each hole of 15% polyacrylamide gel and carries out electrophoresis then.Isolating albumen is eluted on the cellulose nitrate paper with half-dried electric transfer printing, and with the antibody test of anti-human TNF alpha.Use the human TNF alpha sample as standard.

As shown in Figure 4, UV induces TNF α, seen in swimming lane 1.The rapamycin of 2ng/ml suppresses the expression of TNF α, as the finding that significantly reduces of this band in the swimming lane 2.Relative therewith, lipopolysaccharide (LPS) by with the situation that is combined in damage dna not of cell surface membrane receptor CD14 under induce TNF α.With the cell induction TNF α (seeing swimming lane 3) of 1ug/ml LPS processing, and rapamycin does not have influence (comparison of swimming lane 3 and swimming lane 4) to the inductive TNF α of this LPS.

Embodiment 2

Present embodiment has proved that the expression of the inductive TNF α of UV mRNA is suppressed by rapamycin.

For proving this principle, people's cell of use has carried the transgenic by chloramphenicol acetyltransferase (CAT) genomic constitution under the control of tumor necrosis factor (TNF α) promoter.This system has been used to study the stimulation that those can cause TNF α genetic transcription.The CAT gene transcription is easy to detect with a simple zymetology test that forms by the acetylated form of thin layer chromatography chlorine detection mycin.The example of these tests is shown in Fig. 5.

Independent substrate is shown in swimming lane 1, and the background acetylation level that is produced by untreated cell extract is shown in swimming lane 2,6 and 9.The acetylation increase that TNF α promoter control CAT down expresses by fluorescence chloromycetin substrate reflects, this causes moving faster type (from the bottom to the top) in thin layer chromatography is tested.

TNF α is handled as inducing with LPS processing (swimming lane 12) as UV ( swimming lane 3,7 and 10) and non-genomic toxicity by the genotoxicity processing.UV induces TNF α to be subjected to rapamycin to suppress (swimming lane 5), and this proof DNA-protein kinase FRAP is that DNA damage is essential to the signal conduction institute of cytokine TNF alpha gene expression.

Compare rapamycin not influence separately (swimming lane 4 compares with swimming lane 2) with untreated cell.TNF α induces also by wortmannin and suppresses when the 500nM, this is the dosage (swimming lane 8) that suppresses the DNA-protein kinase, also suppressed (swimming lane 11) when the 200nM by D-82041 DEISENHOFEN, this is the dosage that specificity suppresses serine phosphorylation, and the latter is the characteristic phosphorylation type of DNA-protein kinase.These results clearly illustrate that UV is inductive generally all needs DNA-protein kinase, particularly FRAP kinases by the initial expression of TNF α promoter, and relates to the phosphorylation of serine residue.

Relative therewith, non-genomic poisonous substance LPS is to the inhibition that is not subjected to rapamycin shown in the swimming lane 12 and 13 of Fig. 6 of inducing of TNF α.

Generally speaking, present embodiment proved, the approach that is damaged to the TNF alpha transcriptional by UV-DNA needs the DNA-protein kinase of rapamycin sensitivity, and the non-DNA damage incident in cell membrane or other places does not relate to this kinases.

Embodiment 3

This area is well-known, and the approach downstream of FRAP kinase activation is the kinase whose phosphorylation of p70S6K, and the latter is the phosphorylation ribosomal protein again, and changes the translation of genetic transcription thing.Therefore the special activatory a kind of measurement index of the kinase whose UV of FRAP is the phosphorylation of p70S6K.Present embodiment uses this measurement index to prove this activation.

Human keratinized cell is handled with UV radiation or LPS then, and is carried out extracting by the introduction of top (seeing embodiment 1 and materials and methods) with the rapamycin pretreatment of 2ng/ml.With extract electrophoresis on 10% polyacrylamide gel, in the Western trace, use then the special antibody of the serine/threonine phosphorylation form of p70S6K is detected.Control as a kind of sample of going up, this polyacrylamide gel dyes with Coomassie brilliant blue, to determine all albumen of load on each swimming lane.

As shown in Figure 7, the UV radiation has increased p70 ^S6KPhosphorylation ( swimming lane 1 and 2 relatively), this phosphorylation is blocked (swimming lane 3) by the rapamycin pretreatment.Relative therewith, the inductive p70S6K phosphorylation of LPS (swimming lane 4) is comparatively speaking to rapamycin insensitive ( swimming lane 4 and 5).The albumen of load equates on the load contrast band proof gel shown in this figure bottom.

Embodiment 4

Present embodiment has proved the direct activation to the DNA-protein kinase activity of damage or dna breakage.

Human keratinized cell be ATM in the HaCat extract or FRAP by with the antibody of (1) anti-ATM or FRAP and (2) with sepharose 4B link coupled immunoprecipitation anti antibody incubation and immunoprecipitation.

By bonded ATM of centrifugal collection or FRAP, mix derived from the proteic phosphorylated substrate peptide of p53 with a kind of then, in the occasion of FRAP, combine with its little subunit protein FKBP.In some occasion, radiating DNA of phage DNA, UV or rapamycin are joined in the reaction.Add adenosine triphosphate then, with reactant 30 ℃ of incubations 2 hours.Then product is incorporated into an elisa plate, and detects with phosphoserine and the special antibody of phosphothreonine.These antibodies resist by (1) and two of alkali phosphatase coupling and (2) nitrophenols phosphoric acid substrate detects.The yellow color that is produced uses the porous plate monitor to measure by optical density at the 405nm place.

The results are shown in Fig. 8.As shown in FIG., the FRAP phosphorylation of p53 peptide is stimulated by the adding of λ-DNA, and the shorter size of λ-DNA has been represented mammalian DNA with many broken ends (FRAP/FKBP/p53 bar with+DNA bar relatively).But, when the DNA that adds at first with the UV radiation when inducing photoproduct, the activity of FRAP much higher (+DNA bar with+comparison of UV-DNA bar).The enhanced phosphorylation of this UV-DNA can be by eliminating (+rapamycin bar) fully with the common incubation of 2ng/ml rapamycin.Similarly, p53 peptide of ATM phosphorylation (ATM/p53 bar) and phosphorylation thereof are stimulated by the adding of λ-DNA, and the latter's shorter size has been represented dna breakage.

Embodiment 5

Present embodiment shows that the ability that rapamycin reduces the inductive TNF α of UV is dose-dependent.

Use 100J/m ²Uv b radiation with the XPTNF2 cell of inducing the CAT gene expression under the TNF α promoter with the ever-increasing rapamycin incubation of concentration.The inhibition level of the expression under this promoter is shown in Fig. 9.

As shown here, when the level of rapamycin is lower than 2ng/ml, the inhibition effect detection of this DNA-kinases inhibitor less than.Can detect the inhibition effect when 2ng/ml, suppressing effect when dosage improves also increases.

Though introduced here and particular embodiment of the present invention be described, should be appreciated that and under prerequisite without departing from the spirit and scope of the present invention, to make amendment to them.

The content of above-cited various lists of references is introduced in this form with list of references.

Claims (47)

1. the people is handled to suppress because the method for the release of cytokines that contact gene poisonous substance produces, said method comprises and gives said people to be enough to suppress cell because of the dosage of the release of cytokines that contacts the gene poisonous substance and produce with a kind of DNA-kinases inhibitor.

2. the process of claim 1 wherein that the gene poisonous substance is a ultraviolet.

3. the process of claim 1 wherein that the gene poisonous substance is a kind of chemotherapy agents or radiotherapy reagent.

4. the process of claim 1 wherein that the DNA-kinases inhibitor is a rapamycin.

5. the process of claim 1 wherein that the DNA-kinases inhibitor carries out administration with the dosage of the release of at least a cytokine that is enough to suppress to be selected from il-1, interleukin-6, tumor necrosis factor, IL-10 INTERLEUKIN-10 and ICAIU 1.

6. the people is handled to suppress because the method for the release of cytokines that contact gene poisonous substance produces, said method comprises a kind of DNA-kinases inhibitor to be enough to be reduced by at least the active of a kind of DNA-protein kinase and thereby to suppress cell and give said people because of the dosage of the release of cytokines that contacts the gene poisonous substance and produce.

7. the method for claim 6, wherein the gene poisonous substance is a ultraviolet.

8. the method for claim 6, wherein the gene poisonous substance is a kind of chemotherapy agents or radiotherapy reagent.

9. the method for claim 6, wherein the DNA-kinases inhibitor is a rapamycin.

10. the method for claim 6, wherein the DNA-kinases inhibitor carries out administration with the active dosage that is enough to reduce at least a DNA-protein kinase that is selected from DNA-PK, ATM, ATR and FRAP.

11. the method for claim 6, wherein the inhibition of DNA-kinases inhibitor is selected from the release of at least a cytokine of il-1, interleukin-6, tumor necrosis factor, IL-10 INTERLEUKIN-10 and ICAIU 1.

12. modify a kind of method of effect of gene poisonous substance, comprise by regulating the DNA-protein kinase activity and modify the release of cytokines that the DNA damage effect by the gene poisonous substance produces.

13. treat a kind of method of side effect of gene poisonous substance, comprise by regulating the DNA damage effect that the DNA-protein kinase activity reduces release of cytokines and keeps the gene poisonous substance simultaneously.

14. the method for claim 13, wherein side effect be selected from skin carcinoma, erythema, activated viral, inflammation, fever, feel sick, vomiting, headache, feel cold, abnormal pigment, alopecia or its combination.

15. reduce the method for the patient's who is experiencing the transplant rejection treatment at least a side effect, comprising:

(a) give a kind of chemical compound that can suppress transplant rejection with first kind of dosage level; And

(b) give the said chemical compound of another dosage when contact patients gene poisonous substance, said chemical compound is a kind of DNA-kinases inhibitor.

16. the method for claim 15, wherein chemical compound is a rapamycin.

17. reduce the method for the patient's who is experiencing the transplant rejection treatment at least a side effect, comprising:

(a) give first kind of chemical compound that patient can suppress transplant rejection; And

(b) give patient second kind of chemical compound that can suppress transplant rejection when contact patients gene poisonous substance, said second kind of chemical compound is a kind of DNA-kinases inhibitor.

18. the method for claim 17, wherein first kind of chemical compound is Ciclosporin A.

19. the method for claim 17, wherein second kind of chemical compound is rapamycin.

20. carry out the method for transplant rejection treatment, comprise giving a kind of chemical compound that can suppress transplant rejection to the patient of this treatment of needs with the dosage of the release of cytokines that is enough to suppress produce because of contact gene poisonous substance, said chemical compound is a kind of DNA-kinases inhibitor.

21. the method for claim 20, wherein chemical compound is a rapamycin.

22. carry out the method for transplant rejection treatment, comprise giving a kind of chemical compound to be enough to control simultaneously transplant rejection with the dosage that suppresses because of contacting the release of cytokines that the gene poisonous substance produces to the patient of this treatment of needs, said chemical compound is a kind of DNA-kinases inhibitor.

23. the method for claim 22, wherein chemical compound is a rapamycin.

24. carry out the method for transplant rejection treatment, comprise the patient of this treatment of needs to be enough to providing dosage to give first kind of chemical compound transplant rejection control, and giving second kind of chemical compound to the control of transplant rejection with the dosage that suppresses because of contacting the release of cytokines that the gene poisonous substance produces to be enough to provide simultaneously, said second kind of chemical compound is a kind of DNA-kinases inhibitor.

25. the method for claim 24, wherein first kind of chemical compound is Ciclosporin A.

26. the method for claim 24, wherein second kind of chemical compound is rapamycin.

27. strengthen the method for release of cytokines, the tissue or at least a gene poisonous substance of organ that comprise (a) administration of human, and (b) give said tissue or organ with at least a DNA-protein kinase reinforcing agent to be enough to strengthen the dosage that this tissue or organ respond to the release of cytokines of this gene poisonous substance.

28. the method for claim 27, wherein tissue or organ are just standing a kind of inflammation disease.

29. the method for claim 28, wherein tissue or organ are skin, and inflammation disease is a psoriasis, and the gene poisonous substance is selected from coal tar and psoralen+light.

30. monitor the method that a kind of immunosuppressant reagent delivers medicine to patient, said immunosuppressant is the inhibitor of at least a DNA-protein kinase, said method comprises:

(a) obtain a cell sample from patient; And

(b) activity level of said at least a DNA-protein kinase in the working sample.

31. the method for claim 30 comprises the further step of adjusting in dosage, scheme or the pipeline of immunosuppressant at least one according to the mensuration of step (b).

32. the method for claim 30, wherein immunosuppressant is a rapamycin.

33. measure the method for individuality, comprising to the sensitivity of one or more gene poisonous substance:

(a) obtain a cell sample from individuality; And

(b) to the activity level of at least a DNA-protein kinase of said sample determination.

34. measure the method for individuality, comprising to a kind of sensitivity of gene poisonous substance:

(a) obtain a cell sample from individuality; And

(b) to the activity level of said sample determination DNA-protein kinase, wherein the DNA-protein kinase is special to the DNA damage type that said gene poisonous substance produces.

35. in a kind of biological sample, measure the method for DNA-protein kinase activity, comprising:

(a) DNA isolation-protein kinase from this sample;

(b) with isolating kinases simultaneously with the DNA damage of this kinases sensitivity and this kinases can phosphorylation substrate-function; And

(c) level of mensuration substrate phosphorylation, said level is the indication of this kinase activity.

36. the method for claim 35, wherein the use of the level of substrate phosphorylation is a kind of measures the special antibody of this substrate phosphorylation form.

37. contain the compositions of a kind of DNA-kinases inhibitor and a kind of gene poisonous substance.

38. the compositions of claim 37, wherein the gene poisonous substance is a kind of chemotherapy agents.

39. the compositions of claim 37, wherein the gene poisonous substance is a coal tar.

40. contain the compositions of a kind of DNA albumen-inhibitors of kinases and psoralen.

41. contain the compositions of a kind of DNA-kinases inhibitor and immunosuppressant, wherein immunosuppressant is not a kind of DNA-kinases inhibitor.

42. the compositions of claim 41, wherein the DNA-kinases inhibitor is a rapamycin, but not the immunosuppressant of DNA-kinases inhibitor is a Ciclosporin A.

43. contain the gene protection combination of agents thing of a kind of DNA-kinases inhibitor and a kind of non-DNA-kinases inhibitor.

44. the compositions of claim 43, the gene protection reagent of wherein non-DNA-kinases inhibitor is a kind of opacifier.

45. the compositions of claim 43, the gene protection reagent of wherein non-DNA-kinases inhibitor are a kind of DNA repairases.

46. the compositions of claim 43, wherein the DNA-kinases inhibitor is a rapamycin.

47. reduce patient's administration or transmit the method for the side effect of chemotherapy or radiotherapy, comprise giving at least one place in the position of patient's digestive tract, scalp or chemotherapy or radiotherapy administration or transmission with a kind of DNA-kinases inhibitor.

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